For nearly fifty years, alanine transaminase (ALT) has been a widely used clinical diagnostic marker of acute and chronic hepatic injury. Although elevated serum ALT is a common feature of liver damage, it is also observed in other diseases such as obesity, muscle disease, and in apparently healthy people. The cellular mechanisms leading to ALT changes in the above situations are poorly understood, which often renders the interpretation of ALT data problematic. The work proposed in this application is based upon our recent discovery of a novel homologue of human ALT, which we have designated ALT2. ALT2 has significant homology to ALT1 (the only previously known ALT) with 69% identity and 78% similarity at the peptide level. Importantly, the two isoenzymes are encoded by distinct genes and differ significantly in tissue distribution; ALT2 is highly expressed in muscle, kidney and adipose tissue, whereas ALT1 is mainly expressed in liver, kidney and heart. Since current assays to measure serum ALT levels are enzymatic, they do not differentiate between ALT1 and ALT2. Measurement of an isoenzyme in serum may have more diagnostic value than total enzyme activity, as exemplified by creatine kinase-MB for cardiac infarction and bone-specific alkaline phosphatase for bone disease. We hypothesize that ALT2 circulates in serum and contributes variably (depending on disease etiology) to total ALT activity measured in currently available clinical assays. Furthermore, we hypothesize that ALT2 will be particularly elevated in patients with fatty liver and/or obesity, since ALT2, but not ALT1, is abundantly expressed in adipose tissue. To examine these hypotheses, we propose to develop an ALT isoenzyme-specific immunoassay. This assay will be used to measure serum levels of ALT isoenzymes in a host of disease states including liver disease and obesity (stable and during weight loss). We predict that serum ALT2 will be higher in patients with fatty liver or obesity and may be a clinically useful marker for this and other disease states.